Project Summary This study is designed to develop novel sensors for mammalian Autophagy-related protein 8 (mAtg8). Autophagy is an evolutionarily conserved, lysosomal, degradation pathway in which various cytoplasmic components are selectively or non-selectively sequestered by double-membrane vesicle called autophagosomes and transported into lysosomes. This process requires a unique set of proteins called autophagy-related (Atg) proteins, such as Atg8, which play central roles in forming the autophagosomal membrane and recruiting cargo receptors. Malfunction of autophagy has been linked to a wide range of human pathologies, including cancer, different neurodegenerative diseases, immunological disorders, aging, diabetes and heart diseases. Before improvements are enabled for the treatment for diseases caused by autophagy malfunction, extensive efforts must be dedicated to develop robust and reliable methods to monitor, analyze and understand this process. This research proposal will develop original molecular sensors, which recognize Atg8-positive puncta, surpassing current limitations of similar tools available and providing insight into the functions of mAtg8s. The basis of the proposed approach is to engineer short peptides that can detect endogenous mAtg8 proteins without using an Atg8 interaction motif (AIM) or its equivalent, the LC3 interaction region (LIR). In preliminary work, a novel sensor archetype that binds mAtg8s in an AIM/LIR-independent manner has already been generated by us and shown to co-localize with one of the members of mAtg8-protein family. The specific aims of the proposed work are: (1) to modify and optimize the molecular structure architecture of the sensor archetype followed by the verification of accuracy of the subcellular localization in vivo of the newly generated sensors; and (2) to evaluate the effects of sensor(s) generated in Aim 1 on the autophagy pathway from autophagosome formation to its maturation and degradation. Successful completion of the objectives of this research will result in the development of a novel type of molecular sensor for mAtg8-positive puncta (such as autophagosomes) and the function of mAtg8s. The possibility of using an AIM/LIR-independent fluorescent probe for labeling of mAtg8s will open up an exciting opportunity to monitor populations of endogenous mAtg8s in vivo in a time-dependent manner and without having the sensor compete for the binding of important AIM/LIR-containing proteins. Future research efforts will focus on using the sensors for detecting where endogenous mAtg8s reside in addition to autophagosomal membranes, identifying the uncharacterized protein partners of mAtg8s that bind through their AIM/LIR motif at these cellular locations, and developing a peptide-based fluorescent probe for mAtg8 proteins.